Diabetes mellitus is a group of metabolic diseases characterized by chronic hyperglycemia, an increased blood sugar (glucose) levels. Glycated hemoglobin is a generic term referring to a series of minor hemoglobin components that are formed via the attachment of various sugars, most commonly glucose, to the hemoglobin molecule. The most important of these minor hemoglobin components in respect to diabetes is hemoglobin A1c (HbA1c). It is formed by the attachment of glucose to the N-terminal amino acid residue, valine, on one or both β chains of hemoglobin A. The determination of both total hemoglobin and HbA1c is recognized as clinically important in the diagnosis, monitoring, and control of diabetic patients (see e.g., Harrison's Principles of Internal Medicine, 16th ed., McGraw-Hill, New York, 2005).
Optimal therapy of diabetic patients requires also the evaluation of blood/plasma for various other analytes including for example glucose, ketone, microalbumin, creatine, urea and proteinuria markers (see e.g., Reinauer et al. 2002, World Health Organization “Laboratory Diagnosis and Monitoring of Diabetes Mellitus”).
The determination of HbA1c and the percentage thereof in total hemoglobin are used in the measurement of the severity of glucose intolerance in a diabetic patient and in management of diabetes mellitus (Lester, Ann. Clin. Biochem. 26:213-219, (1989); Kennedy et al., Br. Med. Bull. 45:174-190, (1989); Fluckiger et al., J. Chromatogr. 429:279-292, (1988); Goldstein, et al., Clin. Chem. 32:B64-70, (1986); Mortensen, Dan. Med. Bull. 32:309-328, (1985); Goldstein et al., CRC Crit. Rev. Clin. Lab. Sci. 21:187-228, (1984); Peacock, J. Clin. Pathol. 37:841-851, (1984); Miedema et al., Ann. Clin. Biochem. 21:2-15, (1984); Mayer et al., Clin. Chem. Acta 127:147-184, (1983); Gabbay, Med. Clin. North Am. 66:1309-1315, (1982). The concentration of hemoglobin A1c is recognized as being directly related to time-averaged blood glucose levels and is correlated with various stages of metabolic control in diabetic patients.
Normal, non-diabetic patients have a HbA1c level of between about 4-8% of total hemoglobin, while the HbA1c level in diabetic patients may range up to about 15% of total hemoglobin. Hypoglycemic patients correspondingly have a HbA1c level below about 3% of total hemoglobin. Various clinical methods have been developed to provide practical means for assessing the HbA1c level in a sample of a patient's blood, with detection levels of 3-20% HbA1c. Such methods include both electrophoretic and chromatographic techniques. In both electrophoretic and chromatographic methods, standards and control materials having known glycosylated hemoglobin concentrations are necessary in order to assure day-to-day consistency of test results and to provide a reference for calibration of equipment.
Hospital labs and clinics are required to operate in compliance with state and federal regulations. The International Federation of Clinical Chemistry Working Group (IFCC-WG) on HbA1cStandardization has developed reference methods for HbA1c analysis. They have established a laboratory network, which includes two reference methods, i.e., mass spectroscopy and capillary electrophoresis. Each network laboratory uses prepared mixtures of purified Hemoglobin A1c and HbA0 as calibrators. The relationship between HbA1c results from the National Glycohemoglobin Standardization Program (NGSP) network and the IFCC network has been evaluated. The NGSP system was established after the Diabetes Control and Complications Trial (DCCT) study showed the relationship between HbA1c and risks for development and/or progression of diabetes complications. See, e.g., Clinical Chemistry 50: 166-174, 2004; 10.1373/clinchem.2003.024802.
Many insurance and Medicare reimbursement programs require that labs be accredited through organizations such as the College of American Pathologists or the American Proficiency Institute. Such accrediting agencies recommend the use of quality controls to verify that an in vitro diagnostic medical device is operating properly. Diagnostic devices such as compact portable devices intended for satellite and point of care settings (including home use) are also subject to similar regulations, such as for example, the devices described in U.S. Pat. No. 5,580,794 and U.S. Pat. No. 5,837,546, the contents of both of which are incorporated herein in their entireties by reference.
In order to make possible a precise determination of the concentration(s) of analyte(s) in a sample solution, a comparison is made with a calibration curve that has been produced/prepared using standard solutions having known concentrations of analytes. Furthermore, for the precision control of the method of determination and for the calibration of automatic analyzers, it is necessary to use standard solutions with known content. Standard solutions that are used for this purpose preferably contain the measurement parameter to be determined in known concentration. The concentration of the parameter ideally lie in the medically relevant measurement range. The handling of the standard solutions is preferably relatively simple. Such standards should also have a storage stability which is as long as possible for obvious practical reasons. In particular, in the case of compact diagnostic devices, such standard controls should ideally have a suitable room temperature stability. The hitherto known standard solutions for HbA1c determinations do not fulfill several of these prerequisites.
Several instruments currently available combine the measurement of blood pH, glucose, ketone, microalbumin, creatine, urea and markers of proteinuria, gases, electrolytes, as well as various other metabolites in one instrument for a comprehensive testing of the patients tissues. Before such tests can be performed, however, the chemistry analyzer being used must be also be calibrated for these additional analytes to ensure that it is functioning properly. At the present time, laboratories routinely perform multiple calibrations to assess reliability and accuracy as to each individual analyte. These separate steps entail repeated accurate pipetting steps and are costly, time-consuming, labor intensive procedures requiring skilled technicians. There is a definite need for a simple, rapid, accurate assay which can be performed by unskilled users. It is toward the fulfillment of this need that this disclosure is directed.
Additionally, at the present time HbA1c quality controls generally require reconstitution or multiple inversion to resuspend fixed red blood cells. These preparations are not only technique sensitive but also time consuming. Furthermore, prior liquid or reconstituted dry control materials are generally turbid, contain particulate matter, and/or have low shelf lives. Turbidity is generally caused by the presence of lipids or cell stroma. Also, prior controls sometimes contain preservatives in order to enhance shelf life. Such preservatives are generally undesirable because they bind with or dissociate the hemoglobin molecule, or otherwise adversely affect hemoglobin present in the control material. Frequently, reconstitution steps are performed improperly and must be repeated. Some hospital labs are required to perform QC cross over studies when they switch to a new lot, therefore having a product with long shelf life reduces the workload. Additionally, since many new analyzers are portable and have room temperature stable cartridges, they are being located in Point of Care or Physicians' offices. These setting do not have much refrigeration space, so a control with room temperature stability is desirable.
Presently, most hemoglobin controls utilize human or bovine hemoglobin moieties (see e.g., Bionostics FDA 510k, K050961 RNA1C CONTROL FOR NYCOCARD HBA1C). There can be several disadvantages in implementation of such controls. A well recognized problem lies in the difficulty to identify non-hazardous preservatives to bind to the protein that also effectively kill microbes. For example, as disclosed in Fiechtner, U.S. Pat. No. 5,589,393, a method of preparing a refrigerated glycated hemoglobin solution required the use of sodium azide and sodium cyanoborohydride, which are known to be hazardous and dangerous materials. Second, such human or bovine hemoglobin controls are problematic in that glucose naturally binds to the hemoglobin and forms HbA1c. Consequently, analyzer controls that utilize human or bovine hemoglobin cannot be formulated to include both glucose and hemoglobin.
Investigators seeking to overcome these limitations have sought to use modified forms of hemoglobin. Many such efforts have lead to compositions that have proved to have absorption characteristics dissimilar to natural hemoglobin, or which are prone to oxidation and are thus, unstable at room temperature.
Others have devised Hb control solutions relying on relatively large molecular weight Hb peptides. Such controls have been shown to present binding problems, as well as insurmountable room temperature stability issues. For example, a large molecular weight peptide begins to resemble to native molecule. Examples of large molecular weight HB peptides can be found in commercially available blood substitutes such as made available by BioPure Corporation of Boston, Mass., USA, under the name Hemopure® blood substitute (hemoglobin glutamer—250—bovine), or HBOC-201 (cross linked hemoglobin).
Thus, there is a need in the art for Hb standard control solutions that avoid the use of hazardous materials, rely on Hb peptides having a relatively low molecular weight, and which are also stable at room temperature (see e.g., Bionostics FDA 510k, K050100 RNA1C HEMOGLOBIN A1C CONTROL FOR BAYER DCA2000 ANALYZER).
In addition, there is also a need for a comprehensive standard control to provide a single reliable reference suitable for the simultaneous calibration of devices used for the measurement of multiple analytes including glycated hemoglobin. To address the shortcomings of presently available products, such a versatile standard should (a) be based on the inclusion of a hemoglobin peptide moiety that is stable at room temperature and does not interfere with the simultaneous calibration for other analytes recurrently tested in the management of diabetes; and (b) be devoid of hazardous material.